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The amino-terminal module of the C4b-binding protein alpha-chain is crucial for C4b binding and factor I-cofactor function

Härdig, Y ; Hillarp, A LU and Dahlbäck, B LU (1997) In The Biochemical journal 323(Pt 2). p.75-469
Abstract

C4b-binding protein (C4BP) regulates the classical pathway C3-convertase of the complement system. Human C4BP is composed of seven identical subunits (alpha-chains) and one unique one (beta-chain). Both types of chains contain homologous repeats called complement control proteins (CCPs); the alpha-chain contains eight CCPs and the beta-chain three. Each alpha-chain contains a binding site for C4b although the detailed localization of this binding site is not known. We have used three different chimeric proteins, originally designed to localize the protein S-binding site on C4BP, to demonstrate the importance of the amino-terminal part of the alpha-chain for the complement-regulatory functions of C4BP. These recombinant proteins were... (More)

C4b-binding protein (C4BP) regulates the classical pathway C3-convertase of the complement system. Human C4BP is composed of seven identical subunits (alpha-chains) and one unique one (beta-chain). Both types of chains contain homologous repeats called complement control proteins (CCPs); the alpha-chain contains eight CCPs and the beta-chain three. Each alpha-chain contains a binding site for C4b although the detailed localization of this binding site is not known. We have used three different chimeric proteins, originally designed to localize the protein S-binding site on C4BP, to demonstrate the importance of the amino-terminal part of the alpha-chain for the complement-regulatory functions of C4BP. These recombinant proteins were composed of C4BP alpha-chains with one, two or three of the amino-terminal CCPs replaced by corresponding CCPs from the C4BP beta-chain. Furthermore, seven different monoclonal antibodies were raised against C4BP and characterized using the recombinant chimeric proteins. Whereas all three recombinant chimeras bind protein S with the same affinity as plasma-purified C4BP, none of them bound to C4b. Three of the antibodies, which were found to bind to alpha-chain CCP 1 and CCP 2, completely inhibited the binding of plasma-purified C4BP to immobilized C4b. In addition, two of these antibodies totally blocked the factor I-cofactor activity of C4BP in a C4b-degradation assay. The binding site for one of the monoclonal antibodies was also studied using electron microscopy where it was confirmed that this antibody bound to the amino-terminal tip of the alpha-chain. These results show that the amino-terminal CCP of the C4BP alpha-chain (CCP 1) is crucial for the C4b binding and factor I-cofactor activity.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Animals, Antibodies, Monoclonal/metabolism, Anticoagulants/metabolism, Binding, Competitive, Complement C4b/metabolism, Complement Factor I/metabolism, Complement Inactivator Proteins, Epitope Mapping, Glycoproteins, Humans, Mice, Mice, Inbred BALB C, Microscopy, Electron, Models, Molecular, Protein Conformation, Protein S/metabolism, Receptors, Complement/chemistry, Recombinant Fusion Proteins/chemistry, Recombinant Proteins/chemistry
in
The Biochemical journal
volume
323
issue
Pt 2
pages
75 - 469
publisher
Portland Press
external identifiers
  • scopus:0030890121
  • pmid:9163340
ISSN
0264-6021
DOI
10.1042/bj3230469
language
English
LU publication?
yes
id
53de4a40-5d91-4cc8-bfa2-d5c7c6be0256
date added to LUP
2022-08-29 10:30:25
date last changed
2024-02-19 17:46:10
@article{53de4a40-5d91-4cc8-bfa2-d5c7c6be0256,
  abstract     = {{<p>C4b-binding protein (C4BP) regulates the classical pathway C3-convertase of the complement system. Human C4BP is composed of seven identical subunits (alpha-chains) and one unique one (beta-chain). Both types of chains contain homologous repeats called complement control proteins (CCPs); the alpha-chain contains eight CCPs and the beta-chain three. Each alpha-chain contains a binding site for C4b although the detailed localization of this binding site is not known. We have used three different chimeric proteins, originally designed to localize the protein S-binding site on C4BP, to demonstrate the importance of the amino-terminal part of the alpha-chain for the complement-regulatory functions of C4BP. These recombinant proteins were composed of C4BP alpha-chains with one, two or three of the amino-terminal CCPs replaced by corresponding CCPs from the C4BP beta-chain. Furthermore, seven different monoclonal antibodies were raised against C4BP and characterized using the recombinant chimeric proteins. Whereas all three recombinant chimeras bind protein S with the same affinity as plasma-purified C4BP, none of them bound to C4b. Three of the antibodies, which were found to bind to alpha-chain CCP 1 and CCP 2, completely inhibited the binding of plasma-purified C4BP to immobilized C4b. In addition, two of these antibodies totally blocked the factor I-cofactor activity of C4BP in a C4b-degradation assay. The binding site for one of the monoclonal antibodies was also studied using electron microscopy where it was confirmed that this antibody bound to the amino-terminal tip of the alpha-chain. These results show that the amino-terminal CCP of the C4BP alpha-chain (CCP 1) is crucial for the C4b binding and factor I-cofactor activity.</p>}},
  author       = {{Härdig, Y and Hillarp, A and Dahlbäck, B}},
  issn         = {{0264-6021}},
  keywords     = {{Animals; Antibodies, Monoclonal/metabolism; Anticoagulants/metabolism; Binding, Competitive; Complement C4b/metabolism; Complement Factor I/metabolism; Complement Inactivator Proteins; Epitope Mapping; Glycoproteins; Humans; Mice; Mice, Inbred BALB C; Microscopy, Electron; Models, Molecular; Protein Conformation; Protein S/metabolism; Receptors, Complement/chemistry; Recombinant Fusion Proteins/chemistry; Recombinant Proteins/chemistry}},
  language     = {{eng}},
  month        = {{04}},
  number       = {{Pt 2}},
  pages        = {{75--469}},
  publisher    = {{Portland Press}},
  series       = {{The Biochemical journal}},
  title        = {{The amino-terminal module of the C4b-binding protein alpha-chain is crucial for C4b binding and factor I-cofactor function}},
  url          = {{http://dx.doi.org/10.1042/bj3230469}},
  doi          = {{10.1042/bj3230469}},
  volume       = {{323}},
  year         = {{1997}},
}